The mechanisms of LH release and desensitization of the pituitary are still poorly known. We investigated the release of LH by dispersed rat pituitary cells, which were cultured with cytodex beads, packed into columns and superfused. The following results were obtained. 1) LH secretion was rapidly decreased by continuous infusion of 10(-8) M LHRH reaching the control level within 3-4 hours. The desensitization was also observed when the pulse amplitude of LHRH was more than 10(-7) M or when the pulse frequency was more than 4 times per hour. 2) Even after this continuous infusion of 10(-8) M LHRH, pituitary cells responded to 10(-6) M LHRH, 50 mM K+ and 3 mM 8-bromo-cAMP. 3) 3 mM 8-bromo-cAMP and 1 microM forskolin induced a significant increase in LH secretion. 4) 1 microM phorbol myristate acetate (PMA), 40 mu 1-oleoyl-2-acetylglycerol (OAG) and 20 microM Ca2+ ionophore (A-23187) caused a remarkable LH release. 5) 100 microM arachidonic acid (AA) caused a significant increase in LH release. However, the pretreatment with a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA) abolished the LH response to AA. These results indicated that protein kinase A, protein kinase C and the Ca2(+)-AA system were involved in the mechanisms of LH secretion from the pituitary respectively, and the desensitization by LHRH was easily induced when the LHRH pulse frequency and pulse amplitude were not appropriate to pituitary stimulation.